The most unique feature of the enteric nervous system (ENS) is its ability to mediate reflexes int he absence of input from the CNS. One such reflex, which is manifested by isolated segments of gut in vitro, is the peristaltic reflex, a descending propulsive wave of oral contraction and anal relaxation evoked by increases in intraluminal pressure. The ability of the ENS to function autonomously is reflected in a complex internal organization and structure that is more similar to the CNS than to peripheral nerve. Progress has been made in functionally identifying final common excitatory and inhibitory enteric motor neurons to smooth muscle and glands. More recently, we identified intrinsic primary afferent neurons in submucosal ganglia. These neurons project to the myenteric plexus and are excited when mechanical stimuli re applied to the mucosa. This excitation is blocked by antagonists of a subtype of 5-HT receptor (5-HT1p) that we have identified in the bowel and demonstrated, with an anti-idiotypic antibody, to be expressed by the sensory neurons. Since pressure induces enterochromaffin (EC) cells to secrete 5-HT, these data support the hypothesis that pressure or distortion provokes EC cell 5-HT secretion, which activates the mucosal processes of the intrinsic sensory neurons that initiate the peristaltic reflex. We now propose to define the enteric microcircuit responsible for this reflex ant to analyze the role played by 5-HT in its initiation and propagation. Specific questions will be: (1) How does pressure cause the secretion of mucosal 5-HT to activate the peristaltic reflex? (We will study stimulus-secretion coupling in EC cells and the role of the 5-HT transporter [which we recently discovered in crypt epithelial cells] in terminating the mucosal action of 5-H.) (2) Which cells int he myenteric plexus constitute a microcircuit with input from intrinsic sensory neurons and output to smooth muscle? (We will trace the circuit with a recombinant strain of pseudorabies virus that expresses beta-galactosidase.) (3) Do serotonergic interneurons play a critical role in the propagation of the peristaltic reflex? (Serotonergic neurons will be selectively lesioned with 5,7-dihydroxytryptamine and neuronal 5-HT will be selectively depleted with tryptamine; neither agent affects EC cells.) (4) How are the effects of the 5-HT 1p receptor mediated? We will identify which isozymes of PKC are in myenteric neurons then determine whether; (1) they are activated by 5-HT; (ii) 5-HT increases hydrolysis of phosphoinositides; (iii) 5-HT- responsive enteric neurons contain type 2 adenyl cyclase, the form activated by PKC; (iv) activation of PKC increases cAMP; (v) down regulation of PKC modifies the effect of 5-HT on cAMP; (vi) PKC is the critical mediator of 5-HT1p-mediated effects in enteric neurons. These studies will thus identify both the neuronal circuits that mediate the peristaltic reflex and the roles played in it by 5-HY and the 5-HT1p receptor. A better understanding of enteric reflex pathways should help in developing means of treating the extremely common, often disabling, but presently untreatable functional diseases of the bowel. In addition, the undesirable GI side effects of psychoactive drugs that affect 5-HT may be better understand and, if anticipated, avoided.